There are two major aims of this study, both focused on the goal of characterizing key components of the basic myosin motor mechanism. First, it is clear that myosin in the absence of actin can hydrolyze ATP, but then traps the hydrolysis products. It has been hypothesized that there is an escape route (the back door) that opens when myosin binds to actin. If so, Dr. Morris should be able to block the back door with progressively large side chains that alter myosin kinetics, primarily by slowing phosphate release. The interaction of such mutants with actin will provide fundamental insights into the myosin mechanism. Dr. Morris? second aim is to define the structural alterations that generate the two extremes of myosin function. Based on Dr. Morris? preliminary data, he hypothesizes that myosin V, and likely other myosin family members, have kinetics that are fundamentally different from conventional myosin II. These kinetic differences may allow these motors to work either alone or in small numbers. Dr. Morris has preliminary evidence that nonmuscle myosin IIB functions more like myosin V than the myosin II of muscle, and yet it is 85% identical in sequence to conventional smooth muscle myosin. Thus, production of chimeras between smooth and nonmuscle IIB will allow him to define the regions that underlie fundamental changes in the myosin kinetic cycle. Expression of enzymatically active fragments (S1-like and heavy meromyosin-like fragments) of myosin will be accomplished with the baculovirus/SF9 cell system. Functional evaluation of the expressed myosin will include ATPase measurements, determination of enzyme kinetic parameters and in vitro motility (translocation of actin filaments by myosin). Low resolution structures of the recombinant myosins will be obtained via 3D reconstructions of cryo-electron micrographs derived from S1-decorated actin filaments, and high resolution structures will be obtained through X-ray crystallography.